The aim of this application is to isolate potent inhibitors of the lethal factor (LF) metalloprotease of Bacillus anthracis. We will develop a high throughput fluorescence-based assay for LF, starting with the natural target peptide derived from MAP kinase kinase and optimizing the reaction by developing and selecting variants of the natural substrate. The assay will be used to screen combinatorial chemical libraries and "click" chemical libraries. The basic approach of click chemistry is to design two ligands that will become covalently linked upon binding to the substrate. This linkage reaction will be the 1,3-dipolar cyclo-addition of azides and acetylenes. Preliminary work using this approach on acetylcholinesterase has yielded an inhibitor with a KD of 100 fM. Click chemistry will be complemented by traditional combinatorial chemistry. Libraries will be targeted using the structural information available for LF, supplemented by information from point mutations of the substrate. Information from our previous synthesis of metalloprotease inhibitors will also be used.